Array for Rapid Detection of a Microorganism

ABSTRACT

A method for detecting a microorganism or class of microorganisms is provided. More specifically, the method employs an array that contains a plurality of discrete regions (referred to as “addresses”) spaced apart on a solid support in a predetermined pattern. The addresses are selected so that the array provides a distinct spectral response (e.g., pattern of colors) or “fingerprint” for a particular microorganism or class of microorganisms. For example, the array may provide a certain spectral response in the presence of one microorganism or class of microoryanisms (e.g., gram-negative bacteria), but provide a completely different spectral response in the presence of another microorganism or class of microorganisms (e.g., gram-positive bacteria). Detection of the spectral response provided by the array may thus allow for differentiation between microorganisms.

BACKGROUND OF THE INVENTION

The ability to rapidly detect microorganisms is becoming an increasingproblem in a wide variety of industries, includes the medical and foodindustries. For instance, rapid detection of a microorganism in themedical field may be crucial for proper diagnosis and treatment of anillness. Unfortunately, multiple etiologic agents may be responsible fora particular condition, thereby making it difficult to rapidly identifythe cause of the condition. The need for selective identification of thetype of microorganism is important for a variety of reasons. Forexample, the knowledge of which type of microorganism is present maylead one to identify the particular source of contamination and tochoose an appropriate treatment. Most of the current diagnosticprocedures involve culturing the microorganism for identification, aprocess that usually requires several days and often gives negativeresults. Not only is culturing a lengthy process, but certain pathogens(e.g., mycobacteria) are notoriously difficult to grow outside the host.Although “non-culturing” techniques have been developed, they aretypically designed for only a specific pathogen. Thus, several assaysare required to obtain a diagnosis, which are expensive andtime-consuming.

As such, a need currently exists for a technique of rapidly and simplydetecting the presence of microorganisms, and identifying the particulartype of detected microorganism.

SUMMARY OF THE INVENTION

In accordance with one embodiment of the present invention, a method fordetecting a microorganism in a sample is disclosed. The method comprisescontacting the sample with an array, the array comprising a plurality ofindividual array addresses spaced apart in a predetermined pattern on asolid support. The addresses each contain a colorant so that the arrayproduces a visually observable spectral response. The spectral responseis detected (e.g., visually) and correlated to the presence of one ormore microorganisms.

In accordance with another embodiment of the present invention, an arrayfor detecting a microorganism in a sample is disclosed. The arraycomprises a plurality of individual array addresses spaced apart in apredetermined pattern on a solid support. The addresses each contain acolorant so that the array produces a visually observable spectralresponse that is distinct for one or more microorganisms.

Other features and aspects of the present invention are discussed ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, directed to one of ordinary skill in the art, is setforth more particularly in the remainder of the specification, whichmakes reference to the appended figure in which:

FIG. 1 is a perspective view of an exemplary array of the presentinvention prior to contact with a test sample (FIG. 1A), after contactwith a test sample infected with E. coli (FIG. 1B); and after contactwith a test sample infected with S. aureus (FIG. 1C).

Repeat use of reference characters in the present specification anddrawing is intended to represent same or analogous features or elementsof the invention.

DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS

Reference now will be made in detail to various embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, notlimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations may be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, may be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present inventioncovers such modifications and variations as come within the scope of theappended claims and their equivalents.

Generally speaking, the present invention is directed to a method fordetecting a microorganism or class of microorganisms. More specifically,the method employs an array that contains a plurality of discreteregions (referred to as “addresses”) spaced apart on a solid support ina predetermined pattern. The addresses are selected so that the arrayprovides a distinct spectral response (e.g., pattern of colors) or“fingerprint” for a particular microorganism or class of microorganisms.For example, the array may provide a certain spectral response in thepresence of one microorganism or class of microorganisms (e.g.,gram-negative bacteria), but provide a completely different spectralresponse in the presence of another microorganism or class ofmicroorganisms (e.g., gram-positive bacteria). Detection of the spectralresponse provided by the array may thus allow for differentiationbetween microorganisms.

The array addresses contain a colorant capable of exhibiting a colorchange in the presence of one or more microorganisms. That is, thecolorant may change from a first color to a second color, from colorlessto a color, or from a color to colorless. A variety of colorants (e.g.,dyes, pigments, etc.) may be employed in the array of the presentinvention. In one embodiment, for example, pH-sensitive colorants areemployed that are capable of differentiating between certain types ofmicroorganisms. Namely, pH-sensitive colorants can detect a change inthe pH of the growth medium of the microorganism. Bacteria, forinstance, may metabolize the growth medium and generate acidic compounds(e.g., CO₂) or basic compounds (e.g., ammonia) that lead to a change inpH. Likewise, certain microorganisms (e.g., bacteria) contain highlyorganized acid moieties on their cell walls. Because the acidic/basicshift may vary for different microorganisms, pH-sensitive colorants maybe selected in the present invention that are tuned for the desired pHtransition. In this manner, array addresses may be provided withpH-sensitive colorants that are configured to undergo a detectable colorchange only in the presence of microorganisms exhibiting a certainacidic/basic shift.

Phthalein colorants constitute one class of suitable pH-sensitivecolorants that may be employed in the array of the present invention.Phenol Red (i.e., phenolsulfonephthalein), for example, exhibits atransition from yellow to red over the pH range 6.6 to 8.0. Above a pHof about 8.1, Phenol Red turns a bright pink (fuschia) color.Derivatives of Phenol Red may also be suitable for use in the presentinvention, such as those substituted with chloro, bromo, methyl, sodiumcarboxylate, carboxylic acid, hydroxyl and amine functional groups.Exemplary substituted Phenol Red compounds include, for instance,Chlorophenol Red, Metacresol Purple (meta-cresoisulfonephthalein),Cresol Red (ortho-cresolsulfonephthalein), Pyrocatecol Violet(pyrocatecolsulfonephthalein), Chlorophenol Red(3′,3″-dichlorophenolsulfonephthalein), Xylenol Blue (the sodium salt ofpara-xylenolsulfonephthalein), Xylenol Orange, Mordant Blue 3 (C.I.43820), 3,4,5,6-tetrabromophenolsulfonephthalein, Bromoxylenol Blue,Bromophenol Blue (3′,3″,5′,5″-tetrabromophenolsulfonephthalein),Bromochlorophenol Blue (the sodium salt ofdibromo-5′,5″-dichlorophenolsulfonephthalein), Bromocresol Purple(5′,5″-dibromo-ortho-cresolsulfonephthalein), Bromocresol Green(3′,3″,5′,5″-tetrabromo-ortho-cresolsulfonephthalein), and so forth.Still other suitable phthalein colorants are well known in the art, andmay include Bromothymol Blue, Thymol Blue, Bromocresol Purple,thymolphthalein, and phenolphthalein (a common component of universalindicators). For example, Chlorophenol Red exhibits a transition fromyellow to red over a pH range of about 4.8 to 6.4; Bromothymol Blueexhibits a transition from yellow to blue over a pH range of about 6.0to 7.6; thymolphthalein exhibits a transition from colorless to blueover a pH range of about 9.4 to 10.6; phenolphthalein exhibits atransition from colorless to pink over a pH range of about 8.2 to 10.0;Thymol Blue exhibits a first transition from red to yellow over a pHrange of about 1.2 to 2.8 and a second transition from yellow to pH overa pH range of 8.0 to 9.6; Bromophenol Blue exhibits a transition fromyellow to violet over a pH range of about 3.0 to 4.6; Bromocresol Greenexhibits a transition from yellow to blue over a pH range of about 3.8to 5.4; and Bromocresol Purple exhibits a transition from yellow toviolet over a pH of about 5.2 to 6.8.

Hydroxyanthraquinones constitute another suitable class of pH-sensitivecolorants for use in the present invention. Hydroxyanthraquinones havethe following general structure:

The numbers 1-8 shown in the general formula represent a location on thefused ring structure at which substitution of a functional group mayoccur. For hydroxyanthraquinones, at least one of the functional groupsis or contains a hydroxy (—OH) group. Other examples of functionalgroups that may be substituted on the fused ring structure includehalogen groups (e.g., chlorine or bromine groups), sulfonyl groups(e.g., sulfonic acid salts), alkyl groups, benzyl groups, amino groups(e.g., primary, secondary, tertiary, or quaternary amines), carboxygroups, cyano groups, phosphorous groups, etc. Some suitablehydroxyanthraquinones that may be used in the present invention, MordantRed 11 (Alizarin), Mordant Red 3 (Alizarin Red S), Alizarin Yellow R,Alizarin Complexone, Mordant Black 13 (Alizarin Blue Black B), MordantViolet 5 (Alizarin Violet 3R), Alizarin Yellow GG, Natural Red 4(carminic acid), amino-4-hydroxyanthraquinone, Emodin, Nuclear Fast Red,Natural Red 16 (Purpurin), Quinalizarin, and so forth. For instance,carminic acid exhibits a first transition from orange to red over a pHrange of about 3.0 to 5.5 and a second transition from red to purpleover a pH range of about 5.5 to 7.0. Alizarin Yellow R, on the otherhand, exhibits a transition from yellow to orange-red over a pH range ofabout 10.1 to 12.0.

Yet another suitable class of pH-sensitive colorants that may beemployed in the array is aromatic azo compounds having the generalstructure:

X—R₁—N═N—R₂—Y

wherein,

R₁ is an aromatic group;

R₂ is selected from the group consisting of aliphatic and aromaticgroups; and

X and Y are independently selected from the group consisting ofhydrogen, halides, —NO₂, —NH₂, aryl groups, alkyl groups, alkoxy groups,sulfonate groups, —SO₃H, —OH, —COH, —COOH, halides, etc. Also suitableare azo derivatives, such as azoxy compounds (X—R₁—N═NO—R₂—Y) or hydrazocompounds (X—R₁—NH—NH—R₂—Y). Particular examples of such azo compounds(or derivatives thereof) include Methyl Violet, Methyl Yellow, MethylOrange, Methyl Red, and Methyl Green. For instance, Methyl Violetundergoes a transition from yellow to blue-violet at a pH range of about0 to 1.6, Methyl Yellow undergoes a transition from red to yellow at apH range of about 2.9 to 4.0, Methyl Orange undergoes a transition fromred to yellow at a pH range of about 3.1 to 4.4, and Methyl Redundergoes a transition from red to yellow at a pH range of about 4.2 to6.3.

Arylmethanes (e.g., diarylmethanes and triarylmethanes) constitute stillanother class of suitable pH-sensitive colorants for use in the presentinvention. Triarylmethane leuco bases, for example, have the followinggeneral structure:

wherein R, R′, and R″ are independently selected from substituted andunsubstituted aryl groups, such as phenyl, naphthyl, anthracenyl, etc.The aryl groups may be substituted with functional groups, such asamino, hydroxyl, carbonyl, carboxyl, sulfonic, alkyl, and/or other knownfunctional groups. Examples of such triarylmethane leuco bases includeLeucomalachite Green, Pararosaniline Base, Crystal Violet Lactone,Crystal Violet Leuco, Crystal Violet, CI Basic Violet 1, CI Basic Violet2, CI Basic Blue, CI Victoria Blue, N-benzoyl leuco-methylene, etc.Likewise suitable diarylmethane leuco bases may include 4,4′-bis(dimethylamino) benzhydrol (also known as “Michler's hydrol”), Michler'shydrol leucobenzotriazole, Michler's hydrol leucomorpholine, Michler'shydrol leucobenzenesulfonamide, etc. In one particular embodiment, thecolorant is Leucomalachite Green Carbinol (Solvent Green 1) or an analogthereof, which is normally colorless and has the following structure:

Under acidic conditions, one or more free amino groups of theLeucomalachite Green Carbinol form may be protonated to form MalachiteGreen (also known as Aniline Green, Basic Green 4, Diamond Green B, orVictoria Green B), which has the following structure:

Malachite Green typically exhibits a transition from yellow toblue-green over a pH range 0.2 to 1.8. Above a pH of about 1.8,malachite green turns a deep green color.

Still other suitable pH-sensitive colorants that may be employed in thearray include Congo Red, Litmus (azolitmin), Methylene Blue, NeutralRed, Acid Fuchsin, Indigo Carmine, Brilliant Green, Picric acid, MetanilYellow, m-Cresol Purple, Quinaldine Red, Tropaeolin OO,2,6-dinitrophenol, Phioxine B, 2,4-dinitrophenol,4-dimethylaminoazobenzene, 2,5-dinitrophenol, 1-Naphthyl Red,Chlorophenol Red, Hematoxylin, 4-nitrophenol, nitrazine yellow,3-nitrophenol, Alkali Blue, Epsilon Blue, Nile Blue A, universalindicators, and so forth. For instance, Congo Red undergoes a transitionfrom blue to red at a pH range of about 3.0 to 5.2, Litmus undergoes atransition from red to blue at a pH range of about 4.5 to 8.3, andNeutral Red undergoes a transition from red to yellow at a pH range ofabout 11.4 to 13.0.

In addition to pH, other mechanisms may also be wholly or partiallyresponsible for inducing a color change in the colorant. For example,many microorganisms (e.g., bacteria and fungi) produce low molecularweight iron-complexing compounds in growth media, which are known as“siderophores.” Metal complexing colorants may thus be employed in someembodiments of the present invention that undergo a color change in thepresence of siderophores. One particularly suitable class of metalcomplexing colorants are aromatic azo compounds, such as EriochromeBlack T, Eriochrome Blue SE, Eriochrome Blue Black B, Eriochrome CyanineR, Xylenol Orange, Chrome Azurol S, carminic acid, etc. Still othersuitable metal complexing colorants may include Alizarin Complexone,Alizarin S, Arsenazo III, Aurintricarboxylic acid, 2,2′-Bipyidine,Bromopyrogallol Red, Calcon (Eriochrome Blue Black R), Calconcarboxylicacid, Chromotropic acid, disodium salt, Cuprizone,5-(4-Dimethylamino-benzylidene)rhodanine, Dimethylglyoxime,1,5-Diphenylcarbazide, Dithizone, Fluorescein Complexone, Hematoxylin,8-Hydroxyquinoline, 2-Mercaptobenzothiazole, Methylthymol Blue,Murexide, 1-Nitroso-2-naphthol, 2-Nitroso-1-naphthol, Nitroso-R-salt,1,10-Phenanthroline, Phenylfluorone, Phthalein Purple,1-(2-Pyridylazo)-naphthol, 4-(2-Pyridylazo)resorcinol, Pyrogallol Red,Sulfonazo III, 5-Sulfosalicylic acid, 4-(2-Thiazolylazo)resorcinol,Thorin, Thymolthalexon, Tiron, Tolurnr-3,4-dithiol, Zincon, and soforth. It should be noted that one or more of the pH-sensitive colorantsreferenced above may also be classified as metal complexing colorants.

Of course, the colorants need not be capable of independentlydifferentiating between particular microorganisms, so long as theoverall spectral response provided by the array is distinct. In thisregard, colorants may also be employed that exhibit a detectable colorchange in the presence of a broad spectrum of microorganisms.Solvatochromatic colorants, for instance, are believed to exhibit adetectable color change in the presence of a broad spectrum ofmicroorganisms. More specifically, solvatochromatic colorants mayundergo a color change in a certain molecular environment based onsolvent polarity and/or hydrogen bonding propensity. For example, asolvatochromatic colorant may be blue in a polar environment (e.g.,water), but yellow or red in a non-polar environment (e.g., lipid-richsolution). The color produced by the solvatochromatic colorant dependson the molecular polarity difference between the ground and excitedstate of the colorant.

Merocyanine colorants (e.g., mono-, di-, and tri-merocyanines) are oneexample of a type of solvatochromatic colorant that may be employed inthe present invention. Merocyanine colorants, such as merocyanine 540,fall within the donor—simple acceptor colorant classification ofGriffiths as discussed in “Colour and Constitution of Organic Molecules”Academic Press, London (1976). More specifically, merocyanine colorantshave a basic nucleus and acidic nucleus separated by a conjugated chainhaving an even number of methine carbons. Such colorants possess acarbonyl group that acts as an electron acceptor moiety. The electronacceptor is conjugated to an electron donating group, such as a hydroxylor amino group. The merocyanine colorants may be cyclic or acyclic(e.g., vinylalogous amides of cyclic merocyanine colorants). Forexample, cyclic merocyanine colorants generally have the followingstructure:

wherein, n is any integer, including 0. As indicated above by thegeneral structures 1 and 1′, merocyanine colorants typically have acharge separated (i.e., “zwitterionic”) resonance form. Zwitterioniccolorants are those that contain both positive and negative charges andare net neutral, but highly charged. Without intending to be limited bytheory, it is believed that the zwitterionic form contributessignificantly to the ground state of the colorant. The color produced bysuch colorants thus depends on the molecular polarity difference betweenthe ground and excited state of the colorant. One particular example ofa merocyanine colorant that has a ground state more polar than theexcited state is set forth below as structure 2.

The charge-separated left hand canonical 2 is a major contributor to theground state whereas the right hand canonical 2′ is a major contributorto the first excited state. Still other examples of suitable merocyaninecolorants are set forth below in the following structures 3-13.

wherein, “R” is a group, such as methyl, alkyl, aryl, phenyl, etc.

Indigo is another example of a suitable solvatochromatic colorant foruse in the present invention. Indigo has a ground state that issignificantly less polar than the excited state. For example, indigogenerally has the following structure 14:

The left hand canonical form 14 is a major contributor to the groundstate of the colorant, whereas the right hand canonical 14′ is a majorcontributor to the excited state.

Other suitable solvatochromatic colorants that may be used in thepresent invention include those that possess a permanent zwitterionicform. That is, these colorants have formal positive and negative chargescontained within a contiguous π-electron system. Contrary to themerocyanine colorants referenced above, a neutral resonance structurecannot be drawn for such permanent zwitterionic colorants. Exemplarycolorants of this class include N-phenolate betaine colorants, such asthose having the following general structure:

wherein R₁—R₅ are independently selected from the group consisting ofhydrogen, a nitro group (e.g., nitrogen), a halogen, or a linear,branched, or cyclic C₁ to C₂₀ group (e.g., alkyl, phenyl, aryl,pyridinyl, etc.), which may be saturated or unsaturated andunsubstituted or optionally substituted at the same or at differentcarbon atoms with one, two or more halogen, nitro, cyano, hydroxy,alkoxy, amino, phenyl, aryl, pyridinyl, or alkylamino groups. Forexample, the N-phenolate betaine colorant may be4-(2,4,6-triphenylpyridinium-1-yl)-2,6-diphenylphenolate (Reichardt'sdye) having the following general structure 15:

Reichardt's dye shows strong negative solvatochromism and may thusundergo a significant color change from blue to colorless in thepresence of bacteria. That is, Reichardt's dye displays a shift inabsorbance to a shorter wavelength and thus has visible color changes assolvent eluent strength (polarity) increases. Still other examples ofsuitable negatively solvatochromatic pyridinium N-phenolate betainecolorants are set forth below in structures 16-23:

wherein, R is hydrogen, —C(CH₃)₃, —CF₃, or C₆F₁₃.

Still additional examples of colorants having a permanent zwitterionicform include colorants having the following general structure 24:

wherein, n is 0 or greater, and X is oxygen, carbon, nitrogen, sulfur,etc. Particular examples of the permanent zwitterionic colorant shown instructure 24 include the following structures 25-33.

Still other suitable solvatochromatic colorants may include, but are notlimited to 4-dicyanmethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran(DCM); 6-propionyl-2-(dimethylamino)naphthalene (PRODAN);9-(diethylamino)-5H-benzo[a]phenox-azin-5-one (Nile Red);4-(dicyanovinyl)julolidine (DCVJ); phenol blue; stilbazolium colorants;coumarin colorants; ketocyanine colorants; N,N-dimethyl-4-nitroaniline(NDMNA) and N-methyl-2-nitroaniline (NM2NA); Nile blue;1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), anddapoxylbutylsulfonamide (DBS) and other dapoxyl analogs. Besides theabove-mentioned colorants, still other suitable colorants that may beused in the present invention include, but are not limited to,4-[2-N-substituted-1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-1-one,red pyrazolone colorants, azomethine colorants, indoaniline colorants,and mixtures thereof.

Although the above-referenced colorants are classified based on theirmechanism of color change (e.g., pH sensitive, metal complexing, orsolvatochromatic), it should be understood that the present invention isnot limited to any particular mechanism for the color change. Even whena pH-sensitive colorant is employed, for instance, other mechanisms mayactually be wholly or partially responsible for the color change of thecolorant. For example, redox reactions between the colorant andmicroorganism may contribute to the color change.

As stated, the particular selection of colorants is not critical to thepresent invention so long as the array produces a distinct spectralresponse. The individual array addresses may be configured in a varietyof ways to accomplish this purpose. In one particular embodiment,individual array addresses may contain colorants that each exhibits aspectral response in the presence of a microorganism. For example, onearray address may employ a pH-sensitive colorant that undergoes a colorchange at acidic pH levels, while another array address may contain apH-sensitive colorant that undergoes a color change at neutral or basiclevels. Alternatively, the array addresses may simply contain differentchemical classes of colorants, irrespective of the mechanism by whichthey change color. For instance, a first array address may contain aphthaiein colorant, a second array address may contain an aromatic azocolorant, a third array address may contain an anthraquinone colorant,and a fourth array address may contain an arylmethane colorant. Ofcourse, the spectral distinction between individual array addresses neednot always be provided by the use of different colorants. For example,the same colorants may be used in individual array addresses, but at adifferent concentration so as to produce a different spectral response.Certain addresses may likewise contain the same colorant at the sameconcentration, so long as the array as whole is capable of producing adistinct spectral response.

Apart from the composition of the individual array addresses, a varietyof other aspects of the array may be selectively controlled to enhanceits ability to provide a distinct spectral response. One factor thatinfluences the ability of the array to produce a distinct spectralresponse is the number of array addresses employed. Namely, a greaternumber of individual array addresses may enhance the degree that thespectral response varies for different microorganisms. However, anoverly large number of addresses can also lead to difficulty in visuallydifferentiating between spectral responses. Thus, in most embodiments ofthe present invention, the array contains from 2 to 50 array addresses,in some embodiments from 3 to about 40 array addresses, and in someembodiments, from 4 to 20 array addresses. The number of addressesemployed in the array will ultimately depend, at least in part, on thenature of the selected colorants. That is, if the selected colorantshave a similar color change in the presence of a microorganism, a largernumber of addresses may be needed to provide the desired spectralresponse.

Another aspect of the array that may influence its ability to provide adistinctive spectral response is the pattern (e.g., size, spacing,alignment, etc.) of the individual array addresses. The individual arrayaddresses may possess a size effective to permit visual observationwithout unduly increasing the size of the solid support. The width (ordiameter) of the addresses may, for example, range from about 0.01 toabout 100 millimeters, in some embodiments from about 0.1 to about 50millimeters, and in some embodiments, from about 1 to about 20millimeters. The shape of the addresses may also enhance visualobservation of the spectral response. For example, the addresses may bein the form of stripes, bands, dots, or any other geometric shape. Theaddresses may also be spaced apart a certain distance to provide a morevisible spectral response. The spacing between two or more individualarray addresses may, for example, range from about 0.01 to about 100millimeters, in some embodiments from about 0.1 to about 50 millimeters,and in some embodiments, from about 1 to about 20 millimeters. Theoverall pattern of the array may take on virtually any desiredappearance.

One particular embodiment of the array of the present invention is shownin FIG. 1. As depicted in FIG. 1A, for instance, an array 81 is providedthat contains sixteen (16) individual addresses 83 in the form of dotsspaced apart in four (4) separate rows and columns. In this embodiment,each of the addresses 83 includes a colorant. For example, a set offirst addresses 83 a may include colorants that undergo a color changein the presence of E. coli and a set of second addresses 83 b mayinclude colorants that undergo a color change in the presence of S.Aureus. When a sample infected with E. coli contacts the array, thefirst set of addresses 83 a undergo a color change, while the second setof addresses 83 b remains substantially the same or undergo only a faintcolor change (FIG. 1B). When a dermal sample infected with S. aureuscontacts the array 81, the second set of addresses 83 b undergo a colorchange, while the first set of addresses 83 a remains substantially thesame or undergo only a faint color change (FIG. 1C).

The array of the present invention is formed on a solid support, whichis subsequently contacted with the test sample of interest. The solidsupport may be formed from any of a variety materials, such as a film,paper, nonwoven web, knitted fabric, woven fabric, foam, glass, etc. Forexample, the materials used to form the solid support may include, butare not limited to, natural, synthetic, or naturally occurring materialsthat are synthetically modified, such as polysaccharides (e.g.,cellulose materials such as paper and cellulose derivatives, such ascellulose acetate and nitrocellulose); polyether sulfone; polyethylene;nylon; polyvinylidene fluoride (PVDF); polyester; polypropylene; silica;inorganic materials, such as deactivated alumina, diatomaceous earth,MgSO₄, or other inorganic finely divided material uniformly dispersed ina porous polymer matrix, with polymers such as vinyl chloride, vinylchloride-propylene copolymer, and vinyl chloride-vinyl acetatecopolymer; cloth, both naturally occurring (e.g., cotton) and synthetic(e.g., nylon or rayon); porous gels, such as silica gel, agarose,dextran, and gelatin; polymeric films, such as polyacrylamide; and soforth.

Although not required, the colorant may be applied to the solid supportin the form of a composition that contains a mobile carrier. The carriermay be a liquid, gas, gel, etc., and may be selected to provide thedesired performance (time for change of color, contrast betweendifferent areas, and sensitivity) of the colorant. In some embodiments,for instance, the carrier may be an aqueous solvent, such as water, aswell as a non-aqueous solvent, such as glycols (e.g., propylene glycol,butylene glycol, triethylene glycol, hexylene glycol, polyethyleneglycols, ethoxydiglycol, and dipropyleneglycol); alcohols (e.g.,methanol, ethanol, n-propanol, and isopropanol); triglycerides; ethylacetate; acetone; triacetin; acetonitrile, tetrahydrafuran; xylenes;formaldehydes (e.g., dimethylformamide, “DMF”); etc.

Other additives may also be incorporated into the array addresses,either separately or in conjunction with the colorant composition. Inone embodiment, for instance, cyclodextrins are employed that enhancethe sensitivity of the colorant and the contrast between individualarray addresses. While not wishing to be bound by theory, the presentinventors believe that such additives may inhibit the crystallization ofthe colorant and thus provide a more vivid color and also enhancedetection sensitivity. That is, single colorant molecules have greatersensitivity for microorganisms because each colorant molecule is free tointeract with the microbial membrane. In contrast, small crystals ofcolorant have to first dissolve and then penetrate the membrane.Examples of suitable cyclodextrins may include, but are not limited to,hydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin,γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, andhydroxyethyl-γ-cyclodextrin, which are commercially available fromCerestar International of Hammond, Ind.

Surfactants may also help enhance the sensitivity of the colorant andthe contrast between different addresses. Particularly desiredsurfactants are nonionic surfactants, such as ethoxylated alkylphenols,ethoxylated and propoxylated fatty alcohols, ethylene oxide-propyleneoxide block copolymers, ethoxylated esters of fatty (C₈-C₁₈) acids,condensation products of ethylene oxide with long chain amines oramides, condensation products of ethylene oxide with alcohols,acetylenic diols, and mixtures thereof. Various specific examples ofsuitable nonionic surfactants include, but are not limited to, methylgluceth-10, PEG-20 methyl glucose distearate, PEG-20 methyl glucosesesquistearate, C₁₁₋₁₅ pareth-20, ceteth-8, ceteth-12, dodoxynol-12,laureth-15, PEG-20 castor oil, polysorbate 20, steareth-20,polyoxyethylene-10 cetyl ether, polyoxyethylene-10 stearyl ether,polyoxyethylene-20 cetyl ether, polyoxyethylene-10 oleyl ether,polyoxyethylene-20 oleyl ether, an ethoxylated nonylphenol, ethoxylatedoctylphenol, ethoxylated dodecylphenol, or ethoxylated fatty (C₆-C₂₂)alcohol, including 3 to 20 ethylene oxide moieties, polyoxyethylene-20isohexadecyl ether, polyoxyethylene-23 glycerol laurate,polyoxy-ethylene-20 glyceryl stearate, PPG-10 methyl glucose ether,PPG-20 methyl glucose ether, polyoxyethylene-20 sorbitan monoesters,polyoxyethylene-80 castor oil, polyoxyethylene-15 tridecyl ether,polyoxy-ethylene-6 tridecyl ether, laureth-2, laureth-3, laureth-4,PEG-3 castor oil, PEG 600 dioleate, PEG 400 dioleate, and mixturesthereof. Commercially available nonionic surfactants may include theSURFYNOL® range of acetylenic diol surfactants available from AirProducts and Chemicals of Allentown, Pa. and the TWEEN® range ofpolyoxyethylene surfactants available from Fischer Scientific ofPittsburgh, Pa.

A binder may also be employed to facilitate the immobilization of thecolorant on the solid support. For example, water-soluble organicpolymers may be employed as binders, such as polysaccharides andderivatives thereof. Polysaccharides are polymers containing repeatedcarbohydrate units, which may be cationic, anionic, nonionic, and/oramphoteric. In one particular embodiment, the polysaccharide is anonionic, cationic, anionic, and/or amphoteric cellulosic ether.Suitable nonionic cellulosic ethers may include, but are not limited to,alkyl cellulose ethers, such as methyl cellulose and ethyl cellulose;hydroxyalkyl cellulose ethers, such as hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl hydroxybutyl cellulose,hydroxyethyl hydroxypropyl cellulose, hydroxyethyl hydroxybutylcellulose and hydroxyethyl hydroxypropyl hydroxybutyl cellulose; alkylhydroxyalkyl cellulose ethers, such as methyl hydroxyethyl cellulose,methyl hydroxypropyl cellulose, ethyl hydroxyethyl cellulose, ethylhydroxypropyl cellulose, methyl ethyl hydroxyethyl cellulose and methylethyl hydroxypropyl cellulose, and so forth.

Suitable techniques for applying the colorant composition to the solidsupport in the form of individual array addresses include printing,dipping, spraying, melt extruding, coating (e.g., solvent coating,powder coating, brush coating, etc.), spraying, and so forth. Printingtechniques may include, for instance, gravure printing, flexographicprinting, screen printing, laser printing, thermal ribbon printing,piston printing, etc. In one particular embodiment, ink-jet printingtechniques are employed to form the array on the support. Ink-jetprinting is a non-contact printing technique that involves forcing anink through a tiny nozzle (or a series of nozzles) to form droplets thatare directed toward the support. Two techniques are generally utilized,i.e., “DOD” (Drop-On-Demand) or “continuous” ink-jet printing. Incontinuous systems, ink is emitted in a continuous stream under pressurethrough at least one orifice or nozzle. The stream is perturbed by apressurization actuator to break the stream into droplets at a fixeddistance from the orifice. DOD systems, on the other hand, use apressurization actuator at each orifice to break the ink into droplets.The pressurization actuator in each system may be a piezoelectriccrystal, an acoustic device, a thermal device, etc. The selection of thetype of ink jet system varies on the type of material to be printed fromthe print head. For example, conductive materials are sometimes requiredfor continuous systems because the droplets are deflectedelectrostatically. Thus, when the sample channel is formed from adielectric material, DOD printing techniques may be more desirable.

The colorant composition may be formed as a printing ink using any of avariety of known components and/or methods. For example, the printingink may contain water as a carrier, and particularly deionized water.Various co-carriers may also be included in the ink, such as lactam,N-methylpyrrolidone, N-methylacetamide, N-methylmorpholine-N-oxide,N,N-dimethylacetamide, N-methyl formamide,propyleneglycol-monomethylether, tetramethylene sulfone,tripropyleneglycolmonomethylether, propylene glycol, and triethanolamine(TEA). Humectants may also be utilized, such as ethylene glycol;diethylene glycol; glycerine; polyethylene glycol 200, 300, 400, and600; propane 1,3 diol; propylene-glycolmonomethyl ethers, such asDowanol PM (Gallade Chemical Inc., Santa Ana, Calif.); polyhydricalcohols; or combinations thereof. Other additives may also be includedto improve ink performance, such as a chelating agent to sequester metalions that could become involved in chemical reactions over time, acorrosion inhibitor to help protect metal components of the printer orink delivery system, and a surfactant to adjust the ink surface tension.Various other components for use in an ink, such as colorantstabilizers, photoinitiators, binders, surfactants, electrolytic salts,pH adjusters, etc., may be employed as described in U.S. Pat. Nos.5,681,380 to Nohr, et al. and 6,542,379 to Nohr, et al., which areincorporated herein in their entirety by reference thereto for allpurposes.

The exact quantity of a colorant employed within an array address mayvary based on a variety of factors, including the sensitivity of thecolorant, the presence of other additives, the desired degree ofdetectability (e.g., with an unaided eye), the suspected concentrationof the microorganism, etc. In some cases, it is desirable to only detectthe presence of microorganisms at concentrations that are certainthreshold concentrations (e.g., pathogenic). For example, aconcentration of about 1×10³ colony forming units (“CFU”) per milliliterof a test sample or more, in some embodiments about 1×10⁵ CFU/ml ormore, in some embodiments about 1×10⁶ CFU/ml or more, and in someembodiments, about 1×10⁷ CFU/ml or more of a microorganism may bedetected in the present invention. Thus, colorants may be employed in anamount sufficient to undergo a detectable color change in the presenceof a microorganism at a concentration of at least about 1×10³ CFU permilliliter of the test sample. For instance, the colorant may be appliedat a concentration from about 0.1 to about 100 milligrams per milliliterof carrier, in some embodiments from about 0.5 to about 60 milligramsper milliliter of carrier, and in some embodiments, from about 1 toabout 40 milligrams per milliliter of carrier.

The spectral response of the array of the present invention providesinformation regarding the presence of a microorganism to which it isexposed. If desired, the response of a reacted array may be compared(e.g., visually or with the aid of an instrument) to a control array,which is formed in a manner that is the same or similar to the testarray with respect to its responsiveness to microorganisms. Multiplecontrol arrays may likewise be employed that correspond to differenttypes of microorganisms at a certain concentration. Upon comparison, themicroorganism may be identified by selecting the control array having aspectral response that is the same or substantially similar to theresponse of the reacted test array, and then correlating the selectedcontrol array to a particular microorganisms or class of microorganisms.In addition, the array itself may contain one or more colorants that donot generally undergo a detectable color change in the presence of themicroorganism so that the colorant(s) may be used for comparative orcontrol purposes.

The spectral response of the array may be determined either visually orusing instrumentation. In one embodiment, color intensity is measuredwith an optical reader. The actual configuration and structure of theoptical reader may generally vary as is readily understood by thoseskilled in the art. Typically, the optical reader contains anillumination source that is capable of emitting electromagneticradiation and a detector that is capable of registering a signal (e.g.,transmitted or reflected light). The illumination source may be anydevice known in the art that is capable of providing electromagneticradiation, such as light in the visible or near-visible range (e.g.,infrared or ultraviolet light). For example, suitable illuminationsources that may be used in the present invention include, but are notlimited to, light emitting diodes (LED), flashlamps, cold-cathodefluorescent lamps, electroluminescent lamps, and so forth. Theillumination may be multiplexed and/or collimated. In some cases, theillumination may be pulsed to reduce any background interference.Further, illumination may be continuous or may combine continuous wave(CW) and pulsed illumination where multiple illumination beams aremultiplexed (e.g., a pulsed beam is multiplexed with a CW beam),permitting signal discrimination between a signal induced by the CWsource and a signal induced by the pulsed source. For example, in someembodiments, LEDs (e.g., aluminum gallium arsenide red diodes, galliumphosphide green diodes, gallium arsenide phosphide green diodes, orindium gallium nitride violet/blue/ultraviolet (UV) diodes) are used asthe pulsed illumination source. One commercially available example of asuitable UV LED excitation diode suitable for use in the presentinvention is Model NSHU550E (Nichia Corporation), which emits 750 to1000 microwatts of optical power at a forward current of 10 milliamps(3.5-3.9 volts) into a beam with a full-width at half maximum of 10degrees, a peak wavelength of 370-375 nanometers, and a spectralhalf-width of 12 nanometers.

In some cases, the illumination source may provide diffuse illuminationto the colorant. For example, an array of multiple point light sources(e.g., LEDs) may simply be employed to provide relatively diffuseillumination. Another particularly desired illumination source that iscapable of providing diffuse illumination in a relatively inexpensivemanner is an electroluminescent (EL) device. An EL device is generally acapacitor structure that utilizes a luminescent material (e.g., phosphorparticles) sandwiched between electrodes, at least one of which istransparent to allow light to escape. Application of a voltage acrossthe electrodes generates a changing electric field within theluminescent material that causes it to emit light.

The detector may generally be any device known in the art that iscapable of sensing a signal. For instance, the detector may be anelectronic imaging detector that is configured for spatialdiscrimination. Some examples of such electronic imaging sensors includehigh speed, linear charge-coupled devices (CCD), charge-injectiondevices (CID), complementary-metal-oxide-semiconductor (CMOS) devices,and so forth. Such image detectors, for instance, are generallytwo-dimensional arrays of electronic light sensors, although linearimaging detectors (e.g., linear CCD detectors) that include a singleline of detector pixels or light sensors, such as, for example, thoseused for scanning images, may also be used. Each array includes a set ofknown, unique positions that may be referred to as “addresses.” Eachaddress in an image detector is occupied by a sensor that covers an area(e.g., an area typically shaped as a box or a rectangle). This area isgenerally referred to as a “pixel” or pixel area. A detector pixel, forinstance, may be a CCD, CID, or a CMOS sensor, or any other device orsensor that detects or measures light. The size of detector pixels mayvary widely, and may in some cases have a diameter or length as low as0.2 micrometers.

In other embodiments, the detector may be a light sensor that lacksspatial discrimination capabilities. For instance, examples of suchlight sensors may include photomultiplier devices, photodiodes, such asavalanche photodiodes or silicon photodiodes, and so forth. Siliconphotodiodes are sometimes advantageous in that they are inexpensive,sensitive, capable of high-speed operation (short risetime/highbandwidth), and easily integrated into most other semiconductortechnology and monolithic circuitry. In addition, silicon photodiodesare physically small, which enables them to be readily incorporated intovarious types of detection systems. If silicon photodiodes are used,then the wavelength range of the emitted signal may be within theirrange of sensitivity, which is 400 to 1100 nanometers.

Optical readers may generally employ any known detection technique,including, for instance, luminescence (e.g., fluorescence,phosphorescence, etc.), absorbance (e.g., fluorescent ornon-fluorescent), diffraction, etc. In one particular embodiment of thepresent, the optical reader measures color intensity as a function ofabsorbance. In one embodiment, absorbance readings are measured using amicroplate reader from Dynex Technologies of Chantilly, Va. (Model #MRX). In another embodiment, absorbance readings are measured using aconventional test known as “CIELAB”, which is discussed in Pocket Guideto Digital Printing by F. Cost, Delmar Publishers, Albany, N.Y. ISBN0-8273-7592-1 at pages 144 and 145. This method defines three variables,L*, a*, and b*, which correspond to three characteristics of a perceivedcolor based on the opponent theory of color perception. The threevariables have the following meaning:

L*=Lightness (or luminosity), ranging from 0 to 100, where 0=dark and100=light;

a*=Red/green axis, ranging approximately from −100 to 100; positivevalues are reddish and negative values are greenish; and

b*=Yellow/blue axis, ranging approximately from −100 to 100; positivevalues are yellowish and negative values are bluish.

Because CIELAB color space is somewhat visually uniform, a single numbermay be calculated that represents the difference between two colors asperceived by a human. This difference is termed ΔE and calculated bytaking the square root of the sum of the squares of the threedifferences (ΔL*, Δa*, and Δb*) between the two colors. In CIELAB colorspace, each ΔE unit is approximately equal to a “just noticeable”difference between two colors. CIELAB is therefore a good measure for anobjective device-independent color specification system that may be usedas a reference color space for the purpose of color management andexpression of changes in color. Using this test, color intensities (L*,a*, and b*) may thus be measured using, for instance, a handheldspectrophotometer from Minolta Co. Ltd. of Osaka, Japan (Model #CM2600d). This instrument utilizes the D/8 geometry conforming to CIENo. 15, ISO 7724/1, ASTME1164 and JIS Z8722-1982 (diffusedillumination/8-degree viewing system. The D65 light reflected by thespecimen surface at an angle of 8 degrees to the normal of the surfaceis received by the specimen-measuring optical system. Still anothersuitable optical reader is the reflectance spectrophotometer describedin U.S. patent App. Pub. No. 2003/0119202 to Kaylor, et al., which isincorporated herein in its entirety by reference thereto for allpurposes. Likewise, transmission-mode detection systems may also be usedin the present invention.

As a result of the present invention, it has been discovered thatmicroorganism contamination may be detected through the use of a arraythat produces a distinct spectral response for a microorganism or classor microorganisms. The microorganisms that may be detected in accordancewith the present invention are not particularly limited, and may includebacteria, yeast, fungi, mold, protozoa, viruses, etc. Several relevantbacterial groups that may be detected in the present invention include,for instance, gram negative rods (e.g., Entereobacteria); gram negativecurved rods (e.g., vibious, Heliobacter, Campylobacter, etc.); gramnegative cocci (e.g., Neisseria); gram positive rods (e.g., Bacillus,Clostridium, etc.); gram positive cocci (e.g., Staphylococcus,Streptococcus, etc.); obligate intracellular parasites (e.g.,Ricckettsia and Chlamydia); acid fast rods (e.g., Myobacterium,Nocardia, etc.); spirochetes (e.g., Treponema, Borellia, etc.); andmycoplasmas (i.e., tiny bacteria that lack a cell wall). Particularlyrelevant bacteria include E. coli (gram negative rod), Klebsiellapneumonia (gram negative rod), Streptococcus (gram positive cocci),Salmonella choleraesuis (gram negative rod), Staphyloccus aureus (grampositive cocci), and P. aeruginosa (gram negative rod).

In addition to bacteria, other microorganisms of interest include moldsand yeasts (e.g., Candida albicans), which belong to the Fungi kingdom.Zygomycota, for example, is a class of fungi that includes black breadmold and other molds that exhibit a symbiotic relationship with plantsand animals. These molds are capable of fusing and forming tough“zygospores.” Ascomycota is another class of fungi, which includesyeasts, powdery mildews, black and blue-green molds, and some speciesthat cause diseases such as Dutch elm disease, apple scab, and ergot.The life cycle of these fungi combines both sexual and asexualreproduction, and the hyphae are subdivided into porous wails that allowfor passage of the nuclei and cytoplasm. Deuteromycota is another classof fungi that includes a miscellaneous collection of fungi that do notfit easily into the aforementioned classes or the Basidiomycota class(which includes most mushrooms, pore fungi, and puffball fungi).Deuteromycetes include the species that create cheese and penicillin,but also includes disease-causing members such as those that lead toathlete's foot and ringworm.

Regardless, the spectral response of the array of the present inventionis rapid and may be detected within a relatively short period of time.For example, the spectral response may occur in about 20 minutes orless, in some embodiments about 10 minutes or less, in some embodimentsabout 5 minutes or less, in some embodiments about 3 minutes or less,and in some embodiments, from about 10 seconds to about 2 minutes. Inthis manner, the array may provide a “real-time” indication of thepresence or absence of a microorganism or class of microorganisms.

The present invention may be better understood with reference to thefollowing examples.

EXAMPLES Materials Employed

All reagents and solvents were obtained from Sigma-Aldrich ChemicalCompany, Inc. of St. Louis, Mo. unless otherwise noted and were usedwithout further purification. The microorganisms used in the study were:

1. Gram negative (viable)

-   -   Escherichia coli (ATCC #8739) (E. coli)    -   Psuedomonas aeruginosa (ATCC #9027) (P. aeruginosa)    -   Klebsiella pneumoniae (ATCC #4352) (K. pneumoniae)    -   Proteus mirabilis (ATCC #7002) (P. mirabilis)    -   Haemophilus influenzae (ATCC #49247) (H. influenzae)    -   Moraxella lacunata (ATCC #17972) (M. lacunata)

2. Gram positive (viable)

-   -   Staphylococcus aureus (ATCC #6538) (S. aureus)    -   Lactobacillus acidophilus (ATCC #11975) (L. acidophilus)    -   Staphylococcus epidermidis (ATCC #12228) (S. epidermidis)    -   Bacillus subtilis (ATCC #19659) (B. subtilis)    -   Enterococcus faecalis (ATCC #29212) (E. faecalis)    -   Streptococcus pyogenes (ATCC #10782) (S. pyogenes)    -   Streptococcus pneumoniae (ATCC #10015) (S. pneumoniae)

3. Yeast (viable)

-   -   Candida albicans (ATCC #10231) (C. albicans)

4. Mold (viable)

-   -   Aureobasidium pullulans (ATCC #16622) (A. pullulans)    -   Penicillium janthinellum (ATCC #10069) (P. janthinellum)

The colorants used in the study are listed with their molecularstructure in Table 1:

TABLE 1 Exemplary Colorants and Their Corresponding Structure ColorantStructure 4-[(1-Methyl-4(1H)- pyridinylidene)ethylidene]-2,5-cyclohexadien-1-one hydrate

3-Ethyl-2-(2-hydroxy-1- propenyl)benzothiazolium chloride

1-Docosyl-4-(4-hydroxystyryl)pyridinium bromide

N,N-Dimethylindoaniline

Quinalizarin

Merocyanine 540

Eriochrome Blue SE

Phenol Red

Nile Blue A

1-(4-Hydroxyphenyl)-2,4,6- triphenylpyridinium hydroxide inner salthydrate

Azomethine-H monosodium salt hydrate

Indigo carmine

Methylene Violet

Eriochrome Blue Black B

Methylene Blue

Nile Red

Trypan Blue

Safranin O

Crystal Violet

Methyl Orange

Chrome Azurol S

Leucocrystal violet

Leucomalachite Green

Leuco xylene cyanole FF

4,5-Dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate

5-Cyano-2-[3-(5-cyano-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1-ethyl-3-(4-sulfobutyl)-1H- benzimidazolium hydroxide innersalt

Acid Green 25

Bathophenanthrolinedisulfonic acid disodium salt trihydrate

Carminic Acid

Celestine Blue

Hematoxylin

Bromophenol Blue

Bromothymol blue

Rose Bengal

Universal indicator 0-5 Not available Universal indicator 3-10 Notavailable Alizarin Complexone

Alizarin Red S

Purpurin

Alizarin

Emodin

Amino-4-hydroxyanthraquinone

Nuclear Fast Red

Chlorophenol Red

Remazol Brilliant Blue R

Procion Blue HB

Phenolphthalein

Ninhydrin

Nitro blue tetrazolium

Orcein

Celestine blue

Tetra Methyl-para-phenylene diamine (TMPD)

5,10,15,20- Tetrakis(pentafluorophenyl)porphyrin iron(III) chloride

Example 1

Various colorants were tested for their ability to undergo a colorchange in the presence of S. aureus, E. coli, and C. albicansmicroorganisms. The colorants tested were Reichardt's dye,1-Docosyl-4-(4-hydroxystyryl)pyridinium bromide,3-Ethyl-2-(2-hydroxy-1-propenyl)-benzothiazolium chloride,4-[(1-Methyl-4(1H)-pyridinylidene)ethylidene]-2,5-cyclohexadien-1-onehydrate, N,N-Dimethylindoaniline, Quinalizarin, Merocyanine 540,Eriochrome® Blue SE (Plasmocorinth B), Phenol Red, Nile Blue A,1-(4-Hydroxyphenyl)-2,4,6-triphenylpyridinium hydroxide inner salthydrate, Azomethine-H monosodium salt hydrate, Indigo Camine, MethyleneViolet, Eriochrome® Blue Black B, Biebrich scarlet-acid fuchsinsolution, Methylene Blue, Nile Red, Trypan Blue, Safranin O, CrystalViolet, Methyl Orange, and Chrome Azurol S.

Unless otherwise specified, the colorants were dissolved indimethylformamide (DMF). The colorant solutions were then pipetted onto15-cm filter paper (available from VWR International—Catalog No.28306-153) and allowed to dry. The filter paper was sectioned intoquadrants to test four (4) samples—i.e., S. aureus, E. coli, C.albicans, and sterile water. 100 microliters of 10⁷ CFU/mL of S. aureuswas pipetted onto the filter paper in one quadrant, 100 microliters of10⁷ CFU/mL of E. coli was pipetted onto the filter paper in a secondquadrant, 100 microliters of 10⁶ CFU/mL of C. albicans was pipetted ontothe filter paper in a third quadrant, and sterile water was pipetted inthe final quadrant. Color changes in the colorants were observed andrecorded for each of the samples tested. The color was recordedimmediately after the color change to inhibit the fading (or loss ofintensity) of the colors as the samples dried. Table 2 presents theobservations from the experiment.

TABLE 2 Observations of Colorant Color Change (Group 1) Color InitialColor Color Color Change Change w/ Colorant Color Change w/ S. aureusChange w/ E. coli w/ C. albicans sterile water Reichardt's dye BlueColorless Colorless Colorless No change 1-Docosyl-4-(4- Yellow Veryfaint Faint orange Faint orange Very faint hydroxystyryl)pyridiniumorange orange bromide 3-Ethyl-2-(2-hydroxy-1- White/ No change No changeNo change No change propenyl)benzothiazolium cream chloride,4-[(1-Methyl-4(1H)- Bright No change No change No change No changepyridinylidene)ethylidene]- yellow 2,5-cyclohexadien-1-one hydrateN,N-Dimethylindoaniline Grey Faint pink Very faint Very faint pink Nochange pink Quinalizarin Peach Yellow Faint purple Purple No changeMerocyanine 540 Hot pink Light purple Yellowish Deeper Reddish pink pinkyellowish pink Eriochrome Blue Deep Very faint Purple Deep purpleLighter pink SE (Plasmocorinth B) pink purple with dark pink border(dissolution) Phenol Red Yellow Yellow with Orange Deep Green withorange red/orange orange border border Nile Blue A Blue Pink Pink PinkNo change 1-(4-Hydroxyphenyl)-2,4,6- Yellow No change No change Nochange No change triphenylpyridinium hydroxide inner salt hydrateAzomethine-H monosodium Yellow/ Lighter with Lighter with Lighter withLighter with salt hydrate peach deeper deeper deeper border deeperborder border (dissolution) border (dissolution) (dissolution)(dissolution) Indigo Carmine Light Deeper light Deeper light Deeperlight Light blue blue blue blue blue with deeper border (dissolution)Methylene Violet Deep Deeper blue Deeper blue Deeper blue No changeblue/ violet Eriochrome ® Blue Black B Dark Lighter Deep purple Deepblue Darker muddy muddy purple muddy purple purple Biebrich scarlet-acidfuchsin Bright Lighter with Lighter with Lighter with Lighter withsolution red deeper deeper deeper border deeper border border(dissolution) border (dissolution) (dissolution) (dissolution) MethyleneBlue* Bright No change No change No change No change blue Nile RedBright Light pink Light pink Light pink Faint pink purple Trypan Blue*Deep No change No change No change Faintly lighter blue with deeperborder (dissolution) Safranin O Bright Yellowish Yellowish Yellowishwith Pinkish with salmon with salmon with salmon salmon edge salmon edgeedge edge Crystal Violet Deep No change No change No change Faintlylighter blue with deeper border (dissolution) Methyl Orange BrightYellow Yellow Yellow Lighter orange orange with dark orange border(dissolution) Chrome Azurol S Pink Light orange Light yellow Brighteryellow Light pink with dark with dark with dark pink with dark orangepink border border pink border border *Dissolved in water

With the exception of Methyl Orange, Nile Red, and Merocyanine 540, theobserved color change was almost immediate (1 to 2 minutes).

Example 2

Various colorants were tested for their ability to undergo a colorchange in the presence of S. aureus, E. coli and C. albicansmicroorganisms. The colorants tested were Leucocrystal Violet,Leucomalachite Green, Leuco xylene cyanole FF,4,5-Dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate,5-Cyano-2-[3-(5-cyano-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1-ethyl-3-(4-sulfobutyl)-1H-benzimidazoliumhydroxide inner salt, Acid Green 25, Bathophenanthrolinedisulfonic aciddisodium salt trihydrate, Carminic Acid, Celestine Blue, Hematoxylin,Bromophenol Blue, Bromothymol Blue, Rose Bengal, Universal Indicator(0-5), and Universal Indicator (3-10). Unless otherwise specified, thecolorants were dissolved in dimethylformamide (DMF). The VWR filterpaper and colorants were prepared as described in Example 1. Table 3presents the observations from the experiment.

TABLE 3 Observations of Colorant Color Change (Group 2) Initial ColorColor Color Color Change Colorant Color Change w/ S. aureus Change w/ E.coli Change w/ C. albicans w/ sterile water Leucocrystal violet WhiteBlue Blue Blue No change Leucomalachite Green White Green Green Green Nochange Leuco xylene cyanole FF White No change No change No change Nochange 4,5-Dihydroxy-1,3- White No change No change No change No changebenzenedisulfonic acid disodium salt monohydrate*5-Cyano-2-[3-(5-cyano-1,3-diethyl- Bright Dark pink Dark purplish DarkLighter pink 1,3-dihydro-2H-benzimidazol-2- reddish pink greenish pinkwith dark pink ylidene)-1-propenyl]-1-ethyl-3-(4- pink bordersulfobutyl)-1H-benzimidazolium (dissolution) hydroxide inner salt AcidGreen 25 Green Lighter green Lighter green Lighter green Lighter greenwith darker with darker with darker with darker green border greenborder green border green border (dissolution) (dissolution)(dissolution) (dissolution) Bathophenanthrolinedisulfonic White Nochange No change No change No change acid disodium salt trihydrate**Carminic Acid* Reddish Pale purple Purple Dark purple Lighter peachpeach with darker peach border (dissolution) Celestine Blue Dark BlueBlue Blue Blue lavender Hematoxylin Pale No change Light purple Darkerpurple Pale yellow yellow with darker yellow border (dissolution)Bromophenol Blue Bright Dark blue Dark blue Dark blue Lighter yellowYellow with orangeish border (dissolution) Bromothymol Blue YellowLighter Light green Darker green Very light yellow with yellow/whitishdarker yellow with darker border yellow border Rose Bengal Hot pinkDarker pink Purplish pink Reddish pink White with dark pink border(dissolution) Universal Indicator (0-5) Yellowish Yellowish YellowishYellowish Lighter green green blue blue blue with dark green border(dissolution) Universal Indicator (3-10) Peach Pinkish Orange-ish YellowDark peach peach yellow *Dissolved in water **Dissolved in DMF and water

With the exception of Leucocrystal Violet, Leucomalachite Green, andLeuco xylene cyanole FF, the observed color change was almost immediate(1 to 2 minutes).

Example 3

Various colorants were tested for their ability to undergo a colorchange in the presence of S. aureus, E. coli and C. albicansmicroorganisms. The colorants tested were Alizarin Complexone, AlizarinRed S, Purpurin, Alizarin, Emodin, Amino-4-hydroxyanthraquinone, NuclearFast Red, Chlorophenol Red, Remazol Brilliant Blue R, Procion Blue HB,Phenolphthalein, tetraphenylporphine, tetra-o-sulphonic acid, andNinhydrin. Unless otherwise specified, the colorants were dissolved indimethylformamide (DMF). The VWR filter paper and colorants wereprepared as described in Example 1. Table 4 presents the observationsfrom the experiment.

TABLE 4 Observations of Colorant Color Change (Group 3) Color ChangeColor Change Color Change Color Change w/ sterile Colorant Initial Colorw/ S. aureus w/ E. coli w/ C. albicans water Alizarin Complexone YellowBrown Reddish Purple No change purple Alizarin Red S Yellow OrangeishPinkish purple Purple Lighter yellow brown with darker yellow border(dissolution) Purpurin Peachish Darker Reddish pink Deeper reddishYellowish orange peachish pink peach orange Alizarin Butter yellow Nochange Light brown Purplish brown Greenish butter yellow Emodin YellowNo change Faint Deeper Greenish Greenish greenish yellow orange orangeAmino-4- Pink Lighter pink Slightly lighter Faintly lighter Darker pinkhydroxyanthraquinone pink pink Nuclear Fast Red Reddish pink Deeperreddish Yellowish pink Yellowish pink Dark pink pink Chlorophenol RedOrange-ish Brown Deep reddish Deeper reddish Lighter yellow purplepurple orangish yellow with darker border (dissolution) RemazolBrilliant Blue R Bright blue Lighter blue Lighter blue Lighter blueLighter blue with dark blue with dark blue with dark blue with dark blueborder border border border (dissolution) (dissolution) (dissolution)(dissolution) Procion Blue HB Teal green No change No change Faintlydarker Lighter teal teal with darker border (dissolution)Phenolphthalein White No change No change No change No changeTetraphenylporphine, Black Grey with Grey with Grey with Grey withtetra-o-sulphonic acid darker borders darker darker borders darker(dissolution) borders (dissolution) borders (dissolution) (dissolution)Ninhydrin White Deep purple Deep purple Slightly lighter No change deep

The observed color change was almost immediate (1 to 2 minutes).

Example 4

The ability to rapidly detect various gram-positive and gram-negativemicroorganisms utilizing the colorants of Examples 1-3 was demonstrated.Additional colorants were also tested, including Plasmocorinth B, NitroBlue, Alizarin Complexone, Orcein, Tetra Methyl-para-phenylene diamine(TMPD), Nile Red, Eriochrome Blue Black B, Phenol Red, Alizarin Red S,Carminic Acid, Fe(III)C₃, Celestine Blue, Kovac's Reagent, Chrome AzurolS, Universal Indicator 3-10, Methyl Orange, Merocyanine 540, and IronIII Chloride Porphyrin. The gram-positive microorganisms tested were S.aureus, L. acidophilus, S. epidermidis, B. subtilis, and E. faecalis.The gram-negative microorganisms tested were E. coli, P. aeruginosa, K.pneumoniae, and P. mirabilis.

The colorant samples were prepared in a manner similar to Example 1.Unless otherwise specified, the colorants were dissolved indimethylformamide (DMF). Each of the colorant solutions were pipettedonto two separate pieces of VWR filter paper and allowed to dry. Onefilter paper sample with the dried colorant was sectioned into fiveapproximately equal sections to test the five gram-positivemicroorganisms. The other filter paper sample was sectioned intoquadrants to test the four gram negative microorganisms. 100 microlitersof 10⁷ CFU/mL of each microorganism sample was pipetted into theirrespective section of the sample of filter paper. Table 5 presents theobservations from the gram positive microorganisms and Table 6 presentsthe observations from the gram negative microorganisms.

TABLE 5 Color Change Observations for Gram Positive Microorganisms ColorColor Color Initial Change Color Change Color Change Change w/ Change w/Colorant Color w/ B. subtilis w/ S. aureus w/ S. epidermidis E. faecalisL. acidophilus Plasmocorinth B Deep pink Purplish Very faint Deeper pinkReddish Deeper pink purplish pink pink reddish pink Nitro Blue YellowishNo No change No change No change No change Tetrazolium white changeAlizarin Yellow Brownish Lighter Lighter Lighter Brownish Complexone redbrownish red brownish red brownish yellow red Orcein Muddy Light Lightermuddy Darker muddy Darker Darker purple purple purple purple muddy muddypurple purple Tetra Methyl- Bright Colorless Colorless Not tested Nottested Colorless para-phenylene lavender diamine (TMPD)* Nile Red BrightLight pink Light pink Light pink Light pink Light pink purple EriochromeBlue Dark Bluish Lighter muddy Darker muddy Darker Darker Black B Muddypurple purple purple muddy muddy purple purple purple Phenol Red YellowOrange Yellow with Yellow with Yellow Greenish with orange border orangeborder with yellow with yellowish orange orange center border borderAlizarin Red S Yellow Brownish Light brown Light brown Light Light pinkbrown Greenish brown Carminic Acid* Reddish Pale Paler purple Palerpurple Purplish Yellowish peach purple peach peach Fe(III)C₃ White No Nochange Not tested Not tested No change change Celestine Blue Dark BlueBlue Blue Blue Blue lavender Kovac's Reagent Pale White with White withWhite with White with White with yellow greenish greenish greenishgreenish greenish center center and center and center and center and andyellow border yellow border yellow brown border yellow border borderChrome Azurol S Pink Pale Light orange Light Light Light red with yellowwith dark yellowish orange dark red with orange border orange with withdark border reddish dark orange orange border border border UniversalPeach Lighter Lighter peach Lighter peach Lighter Red Indicator 3-10peach with yellow with yellow peach with center center yellow centerMethyl Orange Bright Yellow Yellow Yellow Yellow Yellow orangeMerocyanine 540 Hot pink Light Light purple Light purple Light Lightpurple purple purple Iron III Chloride Light Darker Darker Darker DarkerDarker Porphyrin* mustard mustard mustard mustard mustard mustard yellowyellow yellow yellow yellow yellow *Dissolved in water

TABLE 6 Color Change Observations for Gram Negative Microorganisms ColorInitial Change Color Change Color Change Color Change Colorant Color w/E. coli w/ P. aeruginosa w/ K. pneumoniae w/ P. mirabilis PlasmocorinthB Deep pink Light Deep blue Deep reddish Deep reddish pink purple pinkNitro blue Yellowish No No change No change No change tetrazolium whitechange Alizarin Yellow Purple Deeper purple Brownish Purple Complexonepurple Orcein Muddy Light Dark purple Brownish Darker brownish purplepurple purple purple Tetra Methyl-para- Bright Colorless Dark purpleColorless Colorless phenylene lavender diamine (TMPD)* Nile Red BrightLight pink Light pink Light pink Light pink purple Eriochrome Blue DarkBluish Dark blue Darker purple Darker purple Black B Muddy purple purplePhenol Red Yellow Orange Dark Yellow with Orange red/orange orangeborder Alizarin Red S Yellow Brownish Deep reddish Light brownish Deepreddish purple purple purple purple Carminic Acid* Reddish Blueish Darkpurple Paler Bluish Purple peach purple purple Fe(III)C₃ White No Nochange Not tested No change change Celestine Blue Dark Blue Blue BlueBlue lavender Kovac's Reagent Pale White with White with White withWhite with greenish yellow greenish greenish greenish center center andyellow border center and center and and yellow yellow yellow borderborder border Chrome Azurol S Pink Greenish Bright yellow GreenishGreenish yellow with yellow with dark pink yellow with dark dark pinkborder with dark border pink border pink border Universal IndicatorPeach Lighter Light green Darker peach Lighter peach with 3-10 peachwith yellow yellow center with center yellow center Methyl Orange BrightYellow Yellow Yellow Orange/ orange yellow Merocyanine 540 Hot pinkYellowish Yellowish pink Yellowish pink Yellowish pink pink Iron IIIChloride Mustard Darker Darker Darker mustard Darker mustard yellowPorphyrin* yellow mustard mustard yellow yellow yellow *Dissolved inwater

With the exception of Methyl Orange, Nile Red, TetraMethyl-para-phenylene diamine (TMPD), and Merocyanine 540, the observedcolor change was also most immediate (1 to 2 minutes).

Example 5

The ability to rapidly detect upper respiratory pathogens utilizing agroup of colorants was demonstrated. The colorants tested were AlizarinRed S, Universal Indicator 3-10, Nile Red, Plasmocorinth B, Iron IIIPorphyrin, Eriochrome Blue Black B, Chrome Azurol S, Orcein, AlizarinComplexone, Phenol Red, Carminic Acid, Methyl Orange, and TMPD. Theupper respiratory infection pathogens tested were H. influenzae, M.lacunata, S. pyogenes, S. pneumoniae, A. pullulans, and P. janthinellum.The colorant samples were prepared in a manner similar to Example 1.Unless otherwise specified, the colorants were dissolved indimethylformamide (DMF). Color changes in the colorants were observedand recorded for each of the samples tested. Table 7 presents theobservations from the upper respiratory infection pathogens.

TABLE 7 Color Change for Upper Respiratory Infection Pathogens ColorColor Color Color Color Initial Change w/ Change Change w/ Change w/Change Color change Colorant Color H. influenzae w/ M. lacunata S.pyogenes S. pneumoniae w/ A. pullulans w/ P. janthinellum Alizarin Red SDark Red Brownish Light brown Light brown Bright Bright mustard redbrownish brownish yellow yellow yellow Universal Dark Greenish GreenishBrownish Brownish Darker Darker peach Indicator 3-10 peach yellow yellowyellow yellow peach Nile Red Bright Pink Pink Pink Pink Dark pink Darkpink purple Plasmocorinth B Bright Bluish purple Darker Dark pink Darkpink Lighter Lighter bright pink bluish bright pink purple pink Iron IIIMustard Darker Darker Darker Darker Darker Darker Porphyrin* yellowmustard mustard mustard mustard mustard mustard yellow yellow yellowyellow yellow yellow Eriochrome Grape Dark blue Dark blue Dark Darkgrapish Dark Dark grape Blue Black B grapish pink pink grape ChromeLight Light green Light Brownish Brownish red Light pink Light pinkAzurol S orange with dark red green with red with with dark red withdark with dark red border dark red dark red border red border borderborder border Orcein Muddy Bright purple Bright Bluish Darker LighterLighter purple purple muddy muddy muddy muddy purple purple purplepurple Alizarin Yellow Reddish Purple Brown Brown Yellow YellowComplexone purple Phenol Red Orangish Orangish red Bright red GreenishGreenish Bright Bright yellow yellow yellow yellow yellow CarminicBright Purple Dark Brownish/ Brownish/ Brighter Brighter Acid* peachpurple purplish purplish peach peach peach peach Methyl Dark YellowYellow Yellow Yellow Browinsh Brownish Orange orange yellow yellow TMPD*Yellowish White Purple Not tested Pink Not tested Not tested *Dissolvedin water

With the exception of Methyl Orange, Nile Red, andtetramethyl-para-phenylene diamine (TMPD), the observed color change wasalmost immediate (1 to 2 minutes).

Example 6

Filter paper (available from VWR International) was treated withsolutions of Chrome Azurol, Alizarin Complexone, Plasmocorinth B, andPhenol Red (all dissolved in DMF). The samples were hung dry toevaporate the solvent. Solutions of C. albicans, E. coli, and S. aureuswere diluted in ten-fold dilutions using Trypticase Soybean Broth (TSB)media, and is some cases, sterile water. Concentrations ranged from 10⁸CFU/mL (stock solution) down to 10¹ CFU/mL for both E. coli and S.aureus, and 10⁷ CFU/mL (stock solution) down to 10¹ CFU/mL for C.albicans. TSB and water were used as control solutions. 100 μL aliquotsof each solution were applied to the samples. The color changes aresummarized in Tables 8-12.

TABLE 8 Response to Dilutions of C. albicans in TSB media Initial TSBDye Color 10⁶ CFU/ml 10⁵ CFU/ml 10⁴ CFU/ml 10³ CFU/ml 10² CFU/ml 10¹CFU/ml Control Phenol Red Bright orange Slightly Slightly SlightlySlightly Slightly Dark yellow darker darker darker darker darker orangeorange orange orange orange orange Plasmocorinth B Bright PurplishSlightly Slightly Slightly Slightly Slightly Dark pink blue darkerdarker darker darker darker purplish Purplish Purplish Purplish PurplishPurplish blue blue blue blue blue blue Alizarin Bright Brownish SlightlySlightly Slightly Slightly Slightly Dark Complexone yellow purple darkerdarker darker darker darker Brownish Brownish Brownish Brownish BrownishBrownish purple purple purple purple purple purple Chrome Azurol roseGreenish Slightly Slightly Slightly Slightly Slightly Yellowish yellowdarker darker darker darker darker green Greenish Greenish GreenishGreenish Greenish yellow yellow yellow yellow yellow

TABLE 9 Response to Dilutions of S. aureus in TSB media Initial 10⁸CFU/ml TSB Dye Color (undiluted) 10⁷ CFU/ml 10⁶ CFU/ml 10⁵ CFU/ml 10⁴CFU/ml 10³ CFU/ml 10² CFU/ml Control Phenol Red Bright Bright orangeSlightly Slightly Slightly Slightly Slightly Dark yellow yellow darkerdarker darker darker darker orange orange orange orange orange orangePlasmocorinth Bright Bright Purplish Slightly Slightly Slightly SlightlySlightly Dark B pink purplish blue darker darker darker darker darkerpurplish pink Purplish Purplish Purplish Purplish Purplish blue blueblue blue blue blue Alizarin Bright Light Brownish Slightly SlightlySlightly Slightly Slightly dark Complexone yellow brown purple darkerdarker darker darker darker Brownish Brownish Brownish Brownish BrownishBrownish purple purple purple purple purple purple Chrome rose BrownishGreenish Slightly Slightly Slightly Slightly Slightly Yellowish Azurolyellow yellow darker darker darker darker darker green Greenish GreenishGreenish Greenish Greenish yellow yellow yellow yellow yellow

TABLE 10 Response to Dilutions of S. aureus in water 10⁷ CFU/ml DyeInitial Color (in H₂O) Water Control Phenol Red Bright yellow N/A Lightyellow Plasmocorinth B Bright pink Bright pink Light pink AlizarinComplexone Bright yellow Pale yellow Pale yellow Chrome Azurol roseGreenish Light red-pink red-pink

TABLE 11 Response to Dilutions of E. coli in TSB media Initial 10⁸CFU/ml TSB Dye Color (undiluted) 10⁷ CFU/ml 10⁶ CFU/ml 10⁵ CFU/ml 10⁴CFU/ml 10³ CFU/ml 10² CFU/ml Control Phenol Red Bright Light orangeSlightly Slightly Slightly Slightly Slightly Dark yellow orange darkerdarker darker darker darker orange orange orange orange orange orangePlasmocorinth Bright Pinkish Purplish Slightly Slightly SlightlySlightly Slightly Dark B pink purple blue darker darker darker darkerdarker purplish Purplish Purplish Purplish Purplish Purplish blue blueblue blue blue blue Alizarin Bright Purplish Brownish Slightly SlightlySlightly Slightly Slightly dark Complexone yellow brown purple darkerdarker darker darker darker Brownish Brownish Brownish Brownish BrownishBrownish purple purple purple purple purple purple Chrome Azurol roseLight Greenish Slightly Slightly Slightly Slightly Slightly Yellowishgreen yellow darker darker darker darker darker green Greenish GreenishGreenish Greenish Greenish yellow yellow yellow yellow yellow

TABLE 12 Response to Dilutions of E. coli in water 10⁷ CFU/ml DyeInitial Color (in H₂O) Water Control Phenol Red Bright yellow Orangishyellow Light yellow Plasmocorinth B Bright pink Bright pink Light pinkAlizarin Bright yellow Brownish yellow Pale yellow Complexone ChromeAzurol rose Dark green Light red-pink

Thus, a color change was observed for the microorganisms that wasdifferent than the media alone, although the difference was somewhatmore subtle for the dilute solutions. Without intending to be limited intheory, it is believed that the more subtle difference for the dilutesolutions was due in part to the lack of time given to themicroorganisms to condition the media (the experiment was conductedshortly after dilution). In contrast, the stock solutions containedmicroorganisms that had been in the media for 24 hours.

While the invention has been described in detail with respect to thespecific embodiments thereof, it will be appreciated that those skilledin the art, upon attaining an understanding of the foregoing, mayreadily conceive of alterations to, variations of, and equivalents tothese embodiments. Accordingly, the scope of the present inventionshould be assessed as that of the appended claims and any equivalentsthereto.

1-26. (canceled)
 27. An array for detecting a microorganism in a sample,the array comprising a plurality of individual array addresses spacedapart in a predetermined pattern on a solid support, wherein theaddresses each contain a colorant so in at the array produces a visuallyobservable spectral response that is distinct for one or moremicroorganisms.
 28. The array of claim 27, wherein one or more of theaddresses contain a pH-sensitive colorant.
 29. The array of claim 27,wherein the pH sensitive colorant is a phthalein, hydroxyanthraquinone,arylmethane, aromatic azo, or a derivative thereof.
 30. The array ofclaim 27, wherein one or more of the addresses contain a metalcomplexing colorant.
 31. The array of claim 27, wherein one or more ofthe addresses contain a solvatochromoatic colorant.
 32. The array ofclaim 27, wherein the array contains from 2 to 50 individual arrayaddresses.
 33. The array of claim 27, wherein the array contains from 3to 40 individual array addresses.
 34. The array of claim 27, wherein atleast two of the addresses are spaced apart a distance of from about0.01 to about 100 millimeters.
 35. The array of claim 27, wherein atleast two of the addresses are spaced apart a distance of from about 0.1to about 50 millimeters.
 36. The array of claim 27, wherein the spectralresponse is distinct for one or more microorganisms at a concentrationof about 1×10³ or more colony forming units per milliliter of thesample.
 37. The array of claim 27, wherein the spectral response isdistinct for one or more microorganisms at a concentration of about1×10⁶ or more colony forming units per milliliter of the sample.